Microwave-assisted oxidation of silibinin: A simple and preparative method for the synthesis of improved radical scavengers

Article abstract of DOI:10.1016/j.tetlet.2013.09.035

A new and preparative oxidation of silibinin has been developed to give access to two different silibinin derivatives known for their enhanced antioxidant properties. Conventional heating methods were compared with results obtained from microwave (MW) heating. The base-catalysed oxidation of silibinin under MW heating is a very efficient method for the preparation of 2,3-dehydrosilybin and a related silybin rearrangement product. This latter compound shows enhanced radical scavenging properties. Optimised conditions were used to prepare 2,3-dehydrosilybins A and B from optically pure silybins A and B. An efficient, preparative purification method was also developed to enable isolation of different products in high purity.

Full text of DOI:10.1016/j.tetlet.2013.09.035

Tetrahedron Letters 54 (2013) 6279–6282
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Microwave-assisted oxidation of silibinin: a simple and preparative
method for the synthesis of improved radical scavengers
Giovanni Di Fabio, Valeria Romanucci, Mauro De Nisco, Silvana Pedatella, Cinzia Di Marino,
⇑
Armando Zarrelli
Department of Chemical Sciences, University of Napoli ‘Federico II’, Via Cintia 4, I-80126 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2013
Revised 4 September 2013
Accepted 11 September 2013
Available online 19 September 2013
A new and preparative oxidation of silibinin has been developed to give access to two different silibinin
derivatives known for their enhanced antioxidant properties. Conventional heating methods were com-
pared with results obtained from microwave (MW) heating. The base-catalysed oxidation of silibinin
under MW heating is a very efficient method for the preparation of 2,3-dehydrosilybin and a related sily-
bin rearrangement product. This latter compound shows enhanced radical scavenging properties. Opti-
mised conditions were used to prepare 2,3-dehydrosilybins A and B from optically pure silybins A and
B. An efficient, preparative purification method was also developed to enable isolation of different prod-
ucts in high purity.
Keywords:
Silibinin
Silybin
2,3-Dehydrosilybin
Oxidation
Ó 2013 Elsevier Ltd. All rights reserved.
Microwave assisted reactions
Flavolignans are polyphenolic compounds found abundantly in
edible plants (e.g., fruits and vegetables) that are extremely impor-
tant for human nutrition. Silibinin (Fig. 1) is a prominent compo-
nent (approximately 30%) of the silymarin complex extracted
from milk thistle [Silybum marianum (L.) Gaertn. Carduus marianus
L., Asteraceae]. Silibinin exists as two diastereomers: silybin A and
silybin B. The two isomers occur naturally as a mixture in approx-
imately 1:1 ratio and are epimeric at positions C-700 and C-800 in the
lignan moiety.1 Several reviews have suggested the use of the term
‘Silibinin’ for this mixture to prevent confusion with the pure com-
pounds silybin A (700R, 800R) or silybin B (700S, 800S).2,3
This compound most likely causes the characteristic yellow col-
our of silybin preparations. As it is not easily isolated as a natural
product, 2,3-dehydrosilybin was utterly neglected in studies on
the biological activity of silibinin and silymarin. Therefore, simple
synthetic methods were developed to prepare 2,3-dehydrosily-
bin12,13 and its analogues.14,15The preparation of 2,3-dehydrosily-
bin from silibinin was accomplished by different methods,
including treatment with H2O2 in a solution of NaHCO3 or with
N-methylglucamine.12 Recently, this oxidation was effected by
reaction with pyridine at reflux.16 An alternate approach employed
potassium acetate in DMF at 50 °C.17 An important byproduct ob-
Silibinin has long been recognised for its various pharmacolog-
ical properties.4 It has been shown to exhibit antioxidant, hypocho-
lesterolemic,5 antitumour6–8 cardioprotective, neuroprotective and
antiviral activity.9 Many components of silymarin (Fig. 1) occur as
pairs of diastereomers (silibinin, isosilybin, silychristin) or enanti-
omers (2,3-dehydrosilybin), some of which possess very attractive
pharmacological properties. The oxidation product of silibinin, 2,3-
dehydrosilybin, shows more potent antioxidant activity than its
parent compound. This compound also appeared to be more effec-
tive than silibinin in biological assays comparing their antitumour
and antiproliferative potencies.10 The oxidation product has also
shown positive effects against some skin diseases (e.g., psoriasis
and atopic eczema).11 Extracts from seeds of Silybum marianum
were commonly found to contain traces of 2,3-dehydrosilybin.
tained from alkaline treatment of silibinin is hemiacetal 3
(Scheme 1). This compound was first isolated and characterised
ˇ
by Kren, et al. and was found to be a more potent antioxidant than
either silibinin or 2,3-deydrosilibin. Nevertheless, little is known
about its biological properties, as to date it has been obtained only
as an undesired side product.16
The recent report of base-catalysed oxidation of silibinin, sily-
bin and isosilybin generated wide discussion.18 Different reaction
conditions were described that employed various solvents and
bases. In each case the reaction was carried out in solvents (e.g.,
MeOH or EtOH) in which silibinin shows very limited solubility
(less than 10 mg/mL). Reaction yields never exceeded 50%. Further-
more, 2,3-dehydrosilybin (2) was the only product isolated and
could only be recovered through long and tedious crystallisation
steps. Silibinin is virtually insoluble in nonpolar solvents (DCM,
toluene, hexane, diethyl ether) and is relatively soluble in polar
⇑
Corresponding author. Tel.: +39 081674472; fax: +39 081674393.
E-mail address: zarrelli@unina.it (A. Zarrelli).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.09.035

6280
G. Di Fabio et al. / Tetrahedron Letters 54 (2013) 6279–6282
OCH3
OH
OH
8''
7''
9''
O
D
O
O
O
B
2
3
7
1"
HO
O
C
HO
O
O
1'
2'
A
E
4"
OH
OH
OH
OCH3
OH
4
5
3"
OH
O
OH
Silibinin
Isosilybin
OH
OCH3
OH
O
O
O
HO
O
O
HO
O
O
OH
OH
OH
OH
OCH3
OH
OH
Silychristin
2,3-dehydrosilibyn
Figure 1. Some components of the extract from milk thistle.
solvents. Its solubility increases in the presence of a base due to the
deprotonation and the formation of the phenolate ion.
examined the effect of several solvents in our procedure, including
1,4-dioxane, THF, MeOH, pyridine and DMF. We also explored the
utility of solvent mixtures. We investigated the effect of two bases,
AcOK and Et3N. Reactions were monitored by RP-HPLC21 (Fig. 2) and
quenched by removing the solvent under reduced pressure.
The strength of the bases chosen was a significant consideration
because exposure to strong bases, such as alkali metal hydroxides,
leads to the rapid decomposition of silybin. Reaction yields were
calculated by weighing the products obtained after chromato-
graphic purification over a pre-packed RP-18 column. Products
were eluted with a ternary mixture of CH3OH/CH3CN/H2O contain-
ing increasing proportions of CH3CN.22 The formation of product 3
was not observed under conventional heating. The best reaction
yields were obtained when the solvent was DMF (78%), consistent
with previous reports. Yields from reactions in other solvents never
exceeded 60% (Table 1). The purified product 2 was fully character-
ised by NMR (1H, 13C) and ESI-MS analysis, and these data were
compared with published results to confirm the structure. The pur-
ity of 2 was greater than 97% in all cases, as determined by analyt-
ical HPLC.
We recently took an interest in the oxidation of silibinin (1) to
2,3-dehydrosilybin (2). We also sought to develop a preparative
HPLC method to separate the diastereomers of silibinin19 and to ex-
ploit the enhanced solubility of silibinin in tetrahydrofuran (THF) or
1,4-dioxane solvents in which this metabolite is highly soluble
(>120 mg/mL). It is our view that an efficient, preparative base-cat-
alysed oxidation of silibinin has yet to be developed and that consis-
tently effective purification methods are still required. In the last
year, we focused our research on the use of microwave heating in
conjunction with solvents in which silibinin is readily soluble. In
addition to being energy-efficient, microwaves can also enhance
reaction rates and in many cases improve product yield. The increas-
ing interest in this area is evidenced by the large number of papers
and reviews that have appeared in the literature in recent years.20
In this study, we report new preparative conditions for the oxida-
tion of silibinin and silybin. We explored conventional heating
methods and microwave (MW) heating to promote the reaction.
We have also developed a preparative purification method to isolate
2,3-dehydrosilbin (2) and hemiacetal 3, compounds with remark-
able antioxidant activity that continue to spark much interest. We
The base-catalysed oxidation of silibinin was subsequently car-
ried out under microwave heating. We varied several conditions
OH
O
HO
O
O
O
OH
OH
OCH3
OH
1
Conventional
or
MW heating
OH
OH
O
O
O
OH
HO
O
HO
O
O
O
+
O
OH
OH
OH
OCH3
OH
OCH3
OH
2
3
Scheme 1. Base-catalysed oxidation of Silibinin 1.

G. Di Fabio et al. / Tetrahedron Letters 54 (2013) 6279–6282
6281
Table 2
Base catalysed oxidation of silibinin exploiting the heating promoted by microwave
(MW)
Base
Solvent
Temperature Time (h) Yielda (%)
(3 equiv)
(°C)
[2]
[3]
KOAc
KOAc
—
THF
50
50
60
60
1.5
1.5
2.5
2.5
15
18
25
25
—
—
—
—
THF/H2O (9:1, v/v)
THF/TEA (2:1, v/v)
THF/TEA (1:1, v/v)
—
KOAc
KOAc
KOAc
TEA
KOAc
KOAc
Dioxane/CH3OH (8:2, v/v) 110
0.5
0.5
0.5
0.5
10
0.16
n.r.
n.r.
20
20
35
70
Dioxane/CH3OH (8:2, v/v)
Dioxane/CH3OH (8:2, v/v)
Dioxane/CH3OH (8:2, v/v)
Dioxane/CH3OH (8:2, v/v)
DMF
90
80
80
50
50
5
5
—
15
—
—
—
Pyridine
Pyridine
Pyridine
150
110
100
1.5
3.5
3.5
n.r.
32
28
62
18
Figure 2. A typical HPLC profile of
a
reaction under MW heating: analytical
Phenomenex LUNA RP-18 column (5
eluting with H2O/CH3CN (10 to 100% in 50 min), flow rate 1.0 mL/min, monitored at
254 and 288 nm.
l
m particle size, 10.0 mm ꢀ 250 mm i.d.),
a
See experimental details; n.r.= was observed degradation of the reaction
mixture.
Table 1
Base catalysed oxidation of silibinin exploiting the classical heating method (oil bath)
were purified on a pre-packed RP-18 column, eluting with a ternary
mixture of CH3OH/CH3CN/H2O containing increasing proportions of
CH3CN.24 The purified products 2 and 3 were fully characterised by
NMR (1H, 13C) and ESI MS analysis, and these data were compared
with published results to confirm the structures.12 The purity of
products was greater than 97% in all cases, as determined by analyt-
ical HPLC.
Base
Solvent
Temperature (°C)
Time (h)
Yielda (%)
(3 equiv)
[2]
[3]
KOAc
KOAc
TEA
THF
THF
THF
THF
Reflux
Reflux
Reflux
Reflux
24
48
24
48
25
40
25
40
—
—
—
—
TEA
In conclusion, we have described an efficient new preparative
base-catalysed oxidation of silibinin. The oxidation can be carried
out under conventional or microwave (MW) heating. The MW pro-
cedure in minimum solvent volumes gave rapid access to good
yields of 2,3-dehydrosilybin (2) and hemiacetal 3 (Table 2). The opti-
mised conditions were then used to oxidise optically pure silybins A
and B to 2,3-dehydrosilybins A and B, respectively. These results
show that the advantages of MW heating are shortened reaction
times and the production of hemiacetal 3, in particular when pyri-
dine is used as the solvent A preparative purification method has
also been developed to enable the isolation of 2 and 3 in high purity,
which creates the possibility of large scale preparations of 2 and 3.
Access to significant quantities of these compounds will facilitate
the evaluation of their pharmacological properties, which have not
been explored to date. Having optimised oxidation conditions pro-
moted by microwave (MW) heating, we intend to apply this method
to the synthesis of other silybin analogues with improved solubility
and antioxidant properties.
KOAc
KOAc
TEA
Dioxane
Dioxane
Dioxane
Dioxane
Reflux
Reflux
Reflux
Reflux
24
48
24
48
25
50
25
45
—
—
—
—
TEA
KOAc
—
DMF
Pyridine
80
95
0.5
100
78b
—
60 (51)c
— (<10)c
a
b
c
See experimental details.
See Ref. 18.
See Ref. 17.
(solvent, temperature, time) and all reactions were carried out in a
minimum volume of solvent. As expected, reaction yields (Table 2)
were very low in slightly polar solvents with low dielectric con-
stants, such as THF and 1,4-dioxane (yields 625%).23 Similar results
were obtained using solvent mixtures (see Table 2) composed of
CH3OH and H2O, which have high dielectric constants.
In some cases the harsh reaction conditions led to rapid and sub-
stantial decomposition of starting material and the formation of a
brown and insoluble residue. When the reaction was conducted in
1,4-dioxane: MeOH (8:2, v/v) at 80 °C in the presence of AcOK, we
observed partial degradation of silibinin and the formation of pitchy
and insoluble brown material (ca. 40 wt %). Reactions in DMF or pyr-
idine gave very interesting results. Conducting the reaction in DMF
at 50 °C led to the formation of 2 and 3 in 70% and 15% yield, respec-
tively, within 30 min. When the reaction was carried out in pyridine,
2 and 3 were formed in variable yields depending on the reaction
temperature. At 110 °C, the product distribution favoured formation
of 3 (62%) over 2 (32%). At a slightly lower temperature (100 °C), for-
mation of product 2 (28%) predominated over 3 (18%). All reactions
were monitored by RP-HPLC (Fig. 2) and were quenched by remov-
ing the solvent under reduced pressure. In all cases, the yield of 3
(15–62%) was improved over previously reported yields.17 We sub-
jected pure silybins A and B to the MW oxidation conditions. Heat-
ing in DMF at 50 °C gave 2,3-dehydrosilybins A and B in average
yields of 72%. Conducting the MW oxidation in pyridine at 110 °C
gave good yields of the corresponding hemiacetals. Crude products
Acknowledgments
This study was supported by Università degli Studi di Napoli
‘Federico II’ (Progetto FARO) and AIPRAS Onlus (Associazione Itali-
ana per la Promozione delle Ricerche sull’Ambiente e la Salute
umana). We also thank Tecno BiosÒ for the HPLC facilities.
References and notes
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4. Gazák, R.; Walterová, D.; Kren, V. Curr. Med. Chem. 2007, 14, 315.
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6. Lu, W.; Lin, C.; King, T. D.; Chen, H.; Reynolds, R. C.; Li, Y. Cell. Signal. 2012, 24,
2291. and references therein.
7. Karim, O. B.; Rhee, K.-J.; Liu, G.; Zheng, D.; Huso, D. L BMC Cancer 2013, 13, 157.
and references therein.
8. Kauntz, H.; Bousserouel, S.; Gossé, F.; Raul, F. Apoptosis 2011, 1042, 16. and
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G. Di Fabio et al. / Tetrahedron Letters 54 (2013) 6279–6282
9. Payer, B. A.; Reiberger, T.; Rutter, K.; Beinhardt, S.; Staettermayer, A. F.; Peck-
Radosavljevic, M.; Ferenci, P. J. Clin. Virol. 2010, 49, 131.
CH3CN (10–100% in 50 min), flow rate 1.5 mL/min, monitored at 254 and
288 nm.
10. Huber, A.; Thongphasuk, P.; Erben, G.; Lehmann, W. D.; Tuma, S.; Stremmel,
W.; Chamulitrat, W. Biochim. Bioph. Acta 2008, 1780, 837.
22. General purification procedure: The crude material (1 g) was purified by
chromatography over
a
pre-packed column RP-18 (BiotageÒ KP-C-18-HS
11. Dvorák, Z.; Vrzal, R.; Ulrichová, J. Cell. Biol. Toxicol. 2006, 22, 81.
25 g) on a BiotageÒ Isolera Spektra one eluting with a ternary mixture of
CH3OH/CH3CN/H2O containing increasing proportions of CH3CN (from 4:1:5 to
4:3:3, v/v/v). The flow rate was 25 mL/min.
ˇ
12. Halbach, G.; Trost, W. Arzneim. Forsch. 1974, 24, 866.
13. Pelter, A.; Hänsel, R. Chem. Ber. 1975, 108, 790.
ˇ
ˇ
14. Dzubák, P.; Hajdúch, M.; Gazák, R.; Svobodová, A.; Psotová, J.; Walterová, D.;
23. General procedure for oxidation under conventional heating:
a solution of
ˇ
Sedmera, P.; Kren, V. Bioorg. Med. Chem. 2006, 14, 3793.
silibinin (1.0 g, 2.1 mmol) and base (KOAc or Et3N, 6.22 mmol) in an
1
15. Yang, L. X.; Huang, K. X.; Li, H. B.; Gong, J. X.; Feng, Y. B.; Tao, Q. F.; Wu, Y. H.; Li,
X. K.; Wu, X. M.; Zeng, S.; Spencer, S.; Zhao, Y.; Qu, J. J. Med. Chem. 2009, 52,
7732. and references therein.
appropriate solvent (15 mL for 1,4-dioxane, THF or their mixture; 8 mL for
DMF; 6 mL for pyridine) was heated to reflux. The reaction mixture was
quenched by removal of the solvent in vacuo. The crude product was purified
by chromatography over a pre-packed RP-18 column (see Ref. 23).
ˇ
ˇ
16. Gazák, R.; Svobodová, A.; Psotová, J.; Sedmera, P.; Prikrylová, V.; Walterová, D.;
ˇ
Kren, V. Bioorg. Med. Chem. 2004, 12, 5677.
24. General procedure for oxidation under MW heating: all irradiation experiments
were carried out in a dedicated CEM-Explorer and CEM Discover monomode
microwave apparatus, operating at a frequency of 2.45 GHz with continuous
17. Zarrelli, A.; Sgambato, A.; Petito, V.; De Napoli, L.; Previtera, L.; Di Fabio, G.
Bioorg. Med. Chem. Lett. 2011, 21, 4389.
ˇ
18. Gazák, R.; Trouillas, P.; Biedermann, D.; Fuksovà, K.; Marhol, P.; Kuzma, M.;
irradiation power from 0 to 300 W using the standard maximum power
ˇ
Kren, V. Tetrahedron Lett. 2013, 54, 315.
absorbance level of 300 W. A solution of silibinin 1 (1.0 g, 2.1 mmol) and base
(6.22 mmol) in an appropriate solvent (8 mL for THF, THF/H2O, 1,4-dioxane/
MeOH and 4 mL for pyridine and DMF) was placed in a 10 mL glass tube. The
tube was sealed with a Teflon septum, placed in the microwave cavity and
irradiated. Initial microwave irradiation used the minimum wattage required
to ramp the temperature from room temperature to the desired temperature.
The reaction mixture was held at this temperature for the required time. After
the irradiation period, the reaction vessel was cooled rapidly to ambient
temperature by gas jet cooling.
19. Di Fabio, G.; Romanucci, V.; Di Marino, C.; De Napoli, L.; Zarrelli, A. Planta Med.
2013. http://dx.doi.org/10.1055/s-0032-1328703.
20. Microwaves in Organic Synthesis In Loupy, A., Ed.; Wiley-VCH: Weinheim,
2006. and references cited herein.
21. General procedure for HPLC analysis: HPLC analysis was performed using a
Waters 1525 HPLC equipped with a binary gradient pump and a Waters 2996
Photodiode Array Detector using a Phenomenex LUNA RP18 column (5-
particle size, 10.0 mm ꢀ 250 mm i.d.). The column was eluted with H2O/
lm